Der, essential bacterial GTPase, unique and highly conserved in the prokaryotes, is an attractive target for a novel class of antibiotics. We propose a structure-based approach to develop small molecule inhibitors that block GTP binding site of Der GTPase domain GDI. The structure-based inhibitor design will utilize high resolution X-ray structure of Tm-Der. Natural ligands, GTP and GDP, will be docked into the active site of GD1 domain to model the GTPase active and inactive states. Conformations of GD1 Switch loops I and II in both states will be generated by extensive Monte Carlo search and molecular dymanics simulations. The molecular dynamics ensemble obtained for GD1-GDP complex will be used to derive a dynamic pharmacophore for Der inhibitors, which will be applied for a virtual screening of small molecule databases. Nucleotide-phosphate analogues with modifications specifically designed to interact with the switch loops in the inactive state will be selected and tested for enzymatic and cellular activities. Domain motions and interactions upon ligand binding to the GD1 domain will be studied by molecular dynamics to reveal specific sites on the domain interface that may be targeted with allosteric small molecule inhibitors. The objectives of Phase I are: 1) to obtain models of Der in the active and inactive state by docking the GTP and GDP to the crystal structure of Tm-Der; 2) to derive and compare the dynamic 3D structures of inactive and active forms of Der using molecular dynamic simulations; 3) to determine 3D pharmacophore templates for the Der GTP binding sites; 4) to identify small molecules that match the template, and 5) to test the enzymatic and cellular activities of the selected compounds. The goal of Phase I is to identify Der inhibitor with binding affinity in the 1 to 50 micromolar range. The success of Phase I will be followed by Phase II optimization of initial leads into pre-clinical candidates for bacterial GTPase inhibitors. Commercial applications of the research: Clinical effectiveness of the currently used antibiotics is limited by emergence of drug-resistant bacterial strains, and the screening of new antibiotics against traditional macromolecule biosynthesis targets seems to be exhausted. Selective and orally active Der inhibitors will present a novel class of anti-infective agents with high efficacy and low bacterial resistance that may share a significant segment of the commercial market for antibiotics.